Folding Equilibrium Constants of Telomere G-Quadruplexes in Free

Anal. Chem. 2010, 82, 9469–9475

Folding Equilibrium Constants of Telomere G-Quadruplexes in Free State or Associated with Proteins Determined by Isothermal Differential Hybridization Quan Wang,† Li Ma,‡ Yu-hua Hao,† and Zheng Tan*,† State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P. R. China, and College of Life Sciences, Wuhan University, Wuhan 430072, P. R. China Guanine rich (G-rich) nucleic acids form G-quadruplex structures that are implicated in many biological processes, pharmaceutical applications, and molecular machinery. The folding equilibrium constant (KF) of the G-quadruplex not only determines its stability and competition against duplex formation in genomic DNA but also defines its recognition by proteins and drugs and technical specifications. The KF is most conveniently derived from thermal melting analysis that has so far yielded extremely diversified results for the human telomere G-quadruplex. Melting analysis cannot be used for nucleic acids associated with proteins, thus has difficulty to study how protein association affects the folding equilibrium of G-quadruplex structure. In this work, we established an isothermal differential hybridization (IDH) method that is able to determine the KF of G-quadruplex, either alone or associated with proteins. Using this method, we studied the folding equilibrium of the core sequence G3(T2AG3)3 from vertebrate telomere in K+ and Na+ solutions and how it is affected by proteins associated at its adjacent regions. Our results show that the KF obtained for the free G-quadruplex is within 1 order of magnitude of most of those obtained by melting analysis and protein binding beside a G-quadruplex can dramatically destabilize the G-quadruplex. Nucleic acids with multiple guanine tracks can form a fourstranded intramolecular G-quadruplex structure in the presence of metal ions.1,2 Such sequences have been found to present in many essential regions of the human genome,3-5 such as telomeres,6 promoter of oncogenes,7 an immunoglobulin switch,8 * To whom correspondence should be addressed. Phone: +86 (10) 6480-7259. Fax: +86 (10) 6480-7099. E-mail: [email protected], [email protected]. † Chinese Academy of Sciences. ‡ Wuhan University. (1) Gilbert, D. E.; Feigon, J. Curr. Opin. Struct. Biol. 1999, 9, 305–314. (2) Simonsson, T. Biol. Chem. 2001, 382, 621–628. (3) Catasti, P.; Chen, X.; Mariappan, S. V.; Bradbury, E. M.; Gupta, G. Genetica 1999, 106, 15–36. (4) Huppert, J. L.; Balasubramanian, S. Nucleic Acids Res. 2007, 35, 406–413. (5) Du, Z.; Zhao, Y.; Li, N. Genome Res. 2008, 18, 233–241. (6) Blackburn, E. H. Nature 1991, 350, 569–573. 10.1021/ac102168m  2010 American Chemical Society Published on Web 10/28/2010

and the insulin regulatory9 regions. Bioinformatic analysis has identified up to several hundred thousands of putative quadruplex sequences in the genome as well as in RNA of the human and other species.4,10-17 These sequences are gaining intense attention because of their implication in important biological processes, such as regulation of gene expression, and potential as therapeutic targets against cancer and other diseases.18-29 G-quadruplex also has applications in nanomolecular machinery, in which it is used as driving device operating via its folding/unfolding cycles.30-33 (7) Simonsson, T.; Pecinka, P.; Kubista, M. Nucleic Acids Res. 1998, 26, 1167– 1172. (8) Sen, D.; Gilbert, W. Nature 1988, 334, 364–366. (9) Hammond-Kosack, M. C.; Kilpatrick, M. W.; Docherty, K. J. Mol. Endocrinol. 1993, 10, 121–126. (10) Zhang, R.; Lin, Y.; Zhang, C. T. Nucleic Acids Res. 2008, 36, D372–376. (11) Yadav, V. K.; Abraham, J. K.; Mani, P.; Kulshrestha, R.; Chowdhury, S. Nucleic Acids Res. 2008, 36, D381–385. (12) Verma, A.; Halder, K.; Halder, R.; Yadav, V. K.; Rawal, P.; Thakur, R. K.; Mohd, F.; Sharma, A.; Chowdhury, S. J. Med. Chem. 2008, 51, 5641–5649. (13) Kikin, O.; Zappala, Z.; D’Antonio, L.; Bagga, P. S. Nucleic Acids Res. 2008, 36, D141–148. (14) Hershman, S. G.; Chen, Q.; Lee, J. Y.; Kozak, M. L.; Yue, P.; Wang, L. S.; Johnson, F. B. Nucleic Acids Res. 2008, 36, 144–156. (15) Scaria, V.; Hariharan, M.; Arora, A.; Maiti, S. Nucleic Acids Res. 2006, 34, W683–685. (16) Kostadinov, R.; Malhotra, N.; Viotti, M.; Shine, R.; D’Antonio, L.; Bagga, P. Nucleic Acids Res. 2006, 34, D119–124. (17) Kikin, O.; D’Antonio, L.; Bagga, P. S. Nucleic Acids Res. 2006, 34, W676– 682. (18) Maizels, N. Nat. Struct. Mol. Biol. 2006, 13, 1055–1059. (19) Read, M.; Harrison, R. J.; Romagnoli, B.; Tanious, F. A.; Gowan, S. H.; Reszka, A. P.; Wilson, W. D.; Kelland, L. R.; Neidle, S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844–4849. (20) Jing, N.; Sha, W.; Li, Y.; Xiong, W.; Tweardy, D. J. Curr. Pharm. Des. 2005, 11, 2841–2854. (21) Shay, J. W.; Keith, W. N. Br. J. Cancer 2008, 98, 677–683. (22) Chen, B.; Liang, J.; Tian, X.; Liu, X. Biochemistry (Moscow) 2008, 73, 853– 861. (23) Ou, T. M.; Lu, Y. J.; Tan, J. H.; Huang, Z. S.; Wong, K. Y.; Gu, L. Q. ChemMedChem 2008, 3, 690–713. (24) Zhou, J. L.; Lu, Y. J.; Ou, T. M.; Zhou, J. M.; Huang, Z. S.; Zhu, X. F.; Du, C. J.; Bu, X. Z.; Ma, L.; Gu, L. Q.; Li, Y. M.; Chan, A. S. J. Med. Chem. 2005, 48, 7315–7321. (25) Neidle, S.; Read, M. A. Biopolymers 2000, 56, 195–208. (26) Kerwin, S. M. Curr. Pharm. Des. 2000, 6, 441–478. (27) Hurley, L. H.; Wheelhouse, R. T.; Sun, D.; Kerwin, S. M.; Salazar, M.; Fedoroff, O. Y.; Han, F. X.; Han, H.; Izbicka, E.; Von Hoff, D. D. Pharmacol. Ther. 2000, 85, 141–158. (28) Han, H.; Hurley, L. H. Trends Pharmacol. Sci. 2000, 21, 136–142. (29) Mergny, J. L.; Helene, C. Nat. Med. 1998, 4, 1366–1367. (30) Alberti, P.; Mergny, J. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1569– 1573.

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The folding equilibrium constant KF is an important parameter that quantitatively describes G-quadruplex formation by giving the molar ratio of folded to relaxed molecules at equilibrium. From the biological and pharmaceutical point of view, this parameter determines how a G-quadruplex sequence participates in biochemical processes. To mention a few examples, it not only reflects the stability of the structure but also determines its ability to compete against the formation of duplex DNA if present in the genome; it also determines what structure the sequence intends to form when liberated and how it is likely to be recognized by proteins or drugs. In the applications of molecular machinery, it reflects the technical specifications of such parts involving the G-quadruplex. Therefore the measurement of this parameter will provide quantitative information for understanding the biological roles of the G-quadruplex, designing drugs and molecular devices. The KF of the G-quadruplex is commonly derived from thermal melting analysis, in which the unfolding of G-quadruplex is monitored by changes in spectroscopic signal (ultraviolet (UV), circular dichroism (CD), fluorescence) or heat (differential scanning calorimetry (DSC)). The application and limits of thermodynamic methods on the G-quadruplex have been reviewed.34,35 In general, the KF determination is more accurate at temperatures near the melting temperature (Tm) of the structure.36 G-quadruplexes are extremely stable with Tm far above the physiological temperature (37 °C) at which the unfolded species is in an extremely small amount making its quantification difficult. Some G-quadruplexes even do not show a full melting curve when heated to near 100 °C, making it impossible to derive the KF. Besides, melting of G-quadruplexes often features a pre- and post-transition baseline that is difficult to be distinguished from a possible slow structural transition.34 In addition, their denaturation may not be a simple two-state transition as commonly assumed.37 All these factors compromise accurate determination of KF for G-quadruplexes. The huge disparity in the thermodynamic parameters from different studies on the intramolecular human telomere Gquadruplex has recently been reviewed.35 In these studies, the core or minimum telomere sequences have been used. However, nucleic acids can be bound by proteins in vivo. It is difficult to analyze such structures by thermal melting because of the overlap of more than one heat-induced reactions, i.e., melting of nucleic acid, protein, and nucleic acid/protein complex. To overcome the above-mentioned limitations, we established an isothermal differential hybridization (IDH) method to measure the KF of G-quadruplex. Using the IDH, we measured the KF for the native core sequence of the vertebrate telomere G-rich (31) Xu, Y.; Hirao, Y.; Nishimura, Y.; Sugiyama, H. Bioorg. Med. Chem. 2007, 15, 1275–1279. (32) Alberti, P.; Bourdoncle, A.; Sacca, B.; Lacroix, L.; Mergny, J. L. Org. Biomol. Chem. 2006, 4, 3383–3391. (33) Mills, M.; Mergny, J. L.; Klump, H. H. Arch. Biochem. Biophys. 2008, 474, 8–14. (34) Lane, A. N.; Chaires, J. B.; Gray, R. D.; Trent, J. O. Nucleic Acids Res. 2008, 36, 5482–5515. (35) Chaires, J. B. FEBS J. 2010, 277, 1098–1106. (36) Rachwal, P. A.; Fox, K. R. Methods 2007, 43, 291–301. (37) Ren, J.; Qu, X.; Trent, J. O.; Chaires, J. B. Nucleic Acids Res. 2002, 30, 2307–2315.

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DNA G3(T2AG3)3 in free state or associated with protein to mimic the in vivo situation. Our results show that the Gquadruplex formed by the core sequence has very high KF but is dramatically decreased by associated proteins at its end. This fact suggests that the G-quadruplexes under the in vivo conditions should be much less stable than the free structure in the in vitro studies and protein association/dissociation beside the G-quadruplex structure can significantly affect its folding/unfolding equilibrium. Principle of KF Measurement. The measurement of KF by IDH relies on coupling the structural interconversion between the folded and relaxed states of the G-rich strand to a hybridization reaction with a complementary C-rich strand. For a simple hybridization, in which two complementary oligonucleotides R and C form a duplex D as shown below KD

R + C 798 D

(1)

the reaction satisfies the following when equilibrium is reached: KD )

(R0 - D)(C0 - D) RC ) D D

(2)

where KD is the equilibrium dissociation constant representing the ratio of dissociated to associated molecules, R0 and C0 the initial concentration of R and C before hybridization occurs, R, C, and D the concentration of R, C, and D at equilibrium, respectively. The above equation can be resolved to give

D)

(R0 + C0 + KD) - √(R0 + C0 + KD)2 - 4R0C0 2 (3)

If the R strand can fold into G-quadruplex that is not recognized by the C-rich strand, then the folding/unfolding and the hybridization are coupled as follows: KF

R 798 Q

KD

R + C 798 D

(4)

(5)

where KF is the equilibrium folding constant representing the ratio of folded to relaxed molecules at equilibrium. Thus at equilibrium, we have Q R

(6)

R(C0 - D) RC ) D D

(7)

KF ) KD )

Let R0 be the initial concentration of the G-quadruplex forming strand R before hybridization, then we have

R0 ) Q + R + D

(8)

Manipulation of eqs 6-8 gives KDq )

(R0 - D)(C0 - D) D

(9)

where KDq is defined as KDq ) KD(1 + KF)

(10)

Comparing with eq 2, eq 9 is resolved to give

D)

(R0 + C0 + KDq) - √(R0 + C0 + KDq)2 - 4R0C0 2 (11)

which is the same as eq 3 except that the KD is replaced by KDq that can be regarded, by comparing eqs 2 and 9, as the apparent equilibrium dissociation constant in this situation. The KD and KDq both present the ratio of free monomeric reactants over the duplex product. The formation of G-quadruplex reduces the amount of G-rich strand R available for hybridizing to the C-rich strand C, which lead to a (1 + KF)-fold greater KDq than KD in eq 10. In reaction 4, the folding/unfolding was assumed to be a simple two-state equilibrium, which may not be satisfied for some G-quadruplexes. For the vertebrate telomere G-quadruplex in K+ solution, it has been reported that the process involves an intermediate structure, probably a parallel G-quadruplex.38 In this case, the reactions 4 and 5 become KF2

KF1

R 798 Q2 798 Q1

(12)

KD

R + C 798 D

(13)

where Q1 stands for the final G-quadruplex and Q2 the intermediate structure and KF1 and KF2 the equilibrium constants for the corresponding equilibrium, respectively. At equilibrium, KF1 )

Q1 , Q2

KF2 )

Q2 R

(14)

where Q2 denotes the concentration of the intermediate. These two equations can be manipulated to give Q1 + Q2 ) (1 + KF1)KF2 R

(15)

which will become identical to eq 6 if we let Q1 + Q2 ) Q,

(1 + KF1)KF2 ) KF

(16)

(38) Antonacci, C.; Chaires, J. B.; Sheardy, R. D. Biochemistry 2007, 46, 4654– 4660.

Figure 1. (A) Hybridization schemes for the determination of KF for G3(T2AG3)3 by the isothermal differential hybridization (IDH) method. The folding/unfolding of G3(T2AG3)3 (G21) is coupled with the hybridization to a C-rich strand C18 from which the apparent equilibrium dissociation constant KDq defined in eq 9 can be obtained. The KDq is a function of KD and KF as shown in eq 22. The KD is obtained from a reference hybridization in which the G-rich strand TT-G21 does not form G-quadruplex. The KF is then derived using eq 22. Hybridization is accessed by the quenching of a fluorescent dye FAM attached to the 5′ end of C18. (B) Reliable range of KD and KDq determination by hybridization. Theoretical curves showing normalized fluorescence as a function of G-rich strand concentration R0 were obtained using eq 20 with C0 ) 5 nM, ∆k ) 0.7, and KD values shown beside the curves.

Therefore, eqs 6-11 can also be used to describe the Gquadruplex folding/unfolding equilibrium that involves an intermediate, in which the Q species includes both the final and the intermediate structure and KF represents the molar ratio of the sum of such species over the relaxed form. To measure the KF, two G-rich sequences G21 and TT-G21, i.e., G3(T2AG3)3 and TTG(T2AG3)3, are hybridized with the same fluorescently labeled C-rich strand C18, respectively (Figure 1A). Since the G21 is the wild core telomere sequence that can fold into G-quadruplex, its hybridization with C18 at equilibrium is described by eq 11. The TT-G21 is derived from G21 by mutating the GG at the 5′ end into TT (Figure 1A) to abolish its ability to form G-quadruplex (Figure S1 in the Supporting Information). The duplex formation at equilibrium in this case is described by eq 3. Upon duplex formation, the fluorescent dye attached to the C-rich strand is quenched by its interaction with the nucleobase on the G-rich strand by photoinduced electron transfer between them39 leading to a decrease in fluorescence.39,40 The total fluorescence intensity F is then the sum of the fluorescence from the free and bound C-rich strand, i.e., (39) Torimura, M.; Kurata, S.; Yamada, K.; Yokomaku, T.; Kamagata, Y.; Kanagawa, T.; Kurane, R. Anal. Sci. 2001, 17, 155–160. (40) Vaughn, C. P.; Elenitoba-Johnson, K. S. Am. J. Pathol. 2003, 163, 29–35.


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F ) kfC + kbD ) kf(C0 - D) + kbD ) kfC0 + (kb - kf)D (17) where kf and kb is a multiplicative constant associated with the free and bound C-rich strand that reflects both the photophysical properties of the fluorophore and the sensitivity of fluorometer. In the absence of G-rich strand, the above equation gives the fluorescence of the total C-rich strand without hybridization: F0 ) kfC0

(18)

By normalizing against F0, eq 17 can be rewritten as (kf - kb) D F D )1) 1 - ∆k F0 kf C0 C0

(19)

where ∆k ) (kf - kb)/kf, the fraction of fluorescence quenched upon hybridization. Substituting the D in eq 19 with eqs 3 or 11, the fluorescence in the simple and coupled hybridization at equilibrium is given, respectively, by (R0 + C0 + KD) - √(R0 + C0 + KD)2 - 4R0C0 F ) 1 - ∆k F0 2C0 (20) (R0 + C0 + KDq) - √(R0 + C0 + KDq)2 - 4R0C0 F ) 1 - ∆k F0 2C0 (21) Fitting the data F/F0 with the above expressions by a set of hybridization measurements at different initial G-rich strand concentrations R0 and fixed fluorescent C-rich strand C0, we can obtain both the ∆k and KD or KDq. Our experiments (Figure S2 in the Supporting Information) showed that mutations outside of the hybridization region at the 5′ end of the G-rich strand do not affect KD, which means that the two values of KD (Figure 1A) are equal in the two hybridizations described by eqs 10, 20, and 21. Therefore, when KD and KDq are determined, KF can then be deduced from eq 10 as

KF )

KDq -1 KD

(22)

MATERIALS AND METHODS Oligonucleotides. 5′-Labled oligonucleotides were purchased from TaKaRa Biotechnology (Dalian, China) and unlabeled ones from Invitrogen (Beijing, China). Oligonucleotide concentrations were initially determined from their absorbance at 260 nm and extinction coefficients using the OligoAnalyzer 3.1 from IDT (http://www.idtdna.com). Since the accuracy of concentration determination by UV absorbance can have significant variation41 and each measurement of KF involved three oligonucleotides, further concentration calibrations were carried out by setting the working concentration of the FAM labeled C-rich strand (41) Cavaluzzi, M. J.; Borer, P. N. Nucleic Acids Res. 2004, 32, e13.

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(C18) as reference and titrating it with the two unlabeled G-rich strands (G21 and TT-G21), respectively, in 150 mM Li+ solution at 20 °C. KF Measurement. A measurement of KF was carried out with two sets of hybridizations of C18 with G21 and TT-G21 (Figure 1). FAM labeled C18 (5 nM) was mixed with a series of concentrations of G21 or TT-G21 in TE buffer, pH 7.4, containing 10 mM Tris-HCl, 1 mM EDTA, 50 ng/µL BSA, 150 mM K+, or Na+. After 5 min incubation at 95 °C, the samples were cooled down slowly and maintained at 37 °C overnight until measurement (overnight for convenience, longer incubation did not change the result indicating that equilibrium had been reached). Fluorescence was recorded at 37 °C on a Spex Fluorolog-3 fluorometer (HORIBA Jobin Yvon, France). Excitation was set at 480 nm and emission collected at 520 nm with a 5 nm slit. The fluorescence data in the two hybridizations were fitted together to eq 20 or 21 to extract KD and KDq, respectively, and KF was then derived from eq 22. Since the quenching of the fluorescent dye on the C-rich strand is not affected by mutations at the 5′ end of the G-rich strand (Figure S3 in the Supporting Information), the ∆k was set as a globally fitted parameter for both G21 and TT-G21. This ensures the weak hybridization with the G21 was as accurately fitted as the strong hybridization with the TT-G21. Measurement of KF was also conducted with G21 that is labeled at one or both ends with a digoxin and 46 kDa antidigoxin antibody Fab fragment (Digibind, GlaxoSmithKline ltd, Australia). The Fab fragment was added in a 15/1 molar ratio over DNA to warrant saturation of DNA by the antibody (examined by electrophoresis, results not shown). The preparation of samples and measurements of KF were carried out as described above but without heat denaturation. The hybridizations showed no change after 5 h or longer incubation. RESULTS Requirement for KF Determination. The determination of the KF by the IDH was made possible by the measurement of KD and KDq in two sets of hybridizations at the equilibrium state, in which a fixed concentration of a fluorescently labeled C-rich strand was incubated with a series of concentrations of two G-rich strands, respectively (Figure 1A). Several requirements have to be satisfied for the IDH method. First, theoretical calculations revealed that reliable determination of KD and KDq is limited within the range of approximately 10-10 to 10-6 M (Figure 1B). Therefore, proper mismatches on the C-rich strand were introduced (Figure 1B) to increase the KD and KDq so that they both fall within this range. Second, the G-rich strand should not form intermolecular G-quadruplex. Gel electrophoresis confirmed that the G21 formed intramolecular Gquadruplex and ruled out this concern for both the G21 and TT-G21 at the concentrations used (Figure S1 in the Supporting Information). Third, because of the repetitive nature of the G-rich strand, slippage hybridization may occur and interfere with the designated hybridization. As illustrated in Figure S4A in the Supporting Information, there are a few other potential hybridization modes with 11 or less matching nucleotides besides the designated hybridization of 15 matching nucleotides. The comparison between a 15 and an 11 nucleotide (nt) hybridization

Figure 2. Representative measurement of KF for G-quadruplex of G3(T2AG3)3 in 150 mM K+ (top) and Na+ (bottom) solution. The theoretical curves (solid lines) were obtained by fitting eq 20 to the two sets of hybridizations (solid circles for G21 G3(T2AG3)3, open circles for TT-G21 T2G(T2AG3)3) to extract the KDq and KD, respectively. The KF was calculated as KDq/KD - 1. The averaged KF from three measurements (Table 1) was 807.5 in K+ and 179.8 in Na+ solution.

(Figure S4B in the Supporting Information) indicates that such slippage hybridization is more than 100 times weaker than the designated hybridization, thus we assumed that it could be ignored. Fourthly, because the KD in eq 22 has to be determined by the reference hybridization in which the two guanines at the 5′ end of the wild G-rich strand were mutated to prevent G-quadruplex formation, it is important that such mutations, though outside of the hybridization region, would not affect the KD. This was verified by comparing hybridizations with different mutations. The results in Figure S2 in the Supporting Information show that such hybridizations yielded KD values with little difference. KF determination for the G-Quadruplex Core Sequence. After the requirements were satisfied, measurement of KF was first carried out for the free G-rich telomere core sequence G3(T2AG3)3 using G21 and TT-G21 (Figure 1A) in 150 mM K+ or Na+ solution. The results given in Figure 2 show that the G-quadruplex-forming G21 had a weaker hybridization with the C-rich strand than the TT-G21 that does not form G-quadruplex. The calculations (Table 1) yielded a KF of 807.5 in K+ and 179.8 in Na+ solution for the G3(T2AG3)3 G-quadruplex (Figure 2). The larger KF for the K+ than for the Na+ G-quadruplex is in agreement with the known fact that G-quadruplex is more stable in K+ than in Na+ solution.42 There have been several thermodynamic analysis on the G3(T2AG3)3 and similar sequences in the literature from which the KF can be derived.43-47 (42) Hardin, C. C.; Perry, A. G.; White, K. Biopolymers 2000, 56, 147–194. (43) Mergny, J. L.; Phan, A. T.; Lacroix, L. FEBS Lett. 1998, 435, 74–78. (44) Balagurumoorthy, P.; Brahmachari, S. K. J. Biol. Chem. 1994, 269, 21858– 21869. (45) Li, W.; Wu, P.; Ohmichi, T.; Sugimoto, N. FEBS Lett. 2002, 526, 77–81.

Table 2 shows a comparison of our data with six measurements from the literature. It can be seen that the KF values derived from different studies exhibited extremely large discrepancy. The KF we obtained for the K+ G-quadruplex is within 1 order of magnitude of four values from the literatures43,44,46 but differs significantly from two others.45,47 For the Na+ G-quadruplex, ours is also within 1 order of magnitude of the values from four studies43,44,47 but differs significantly from two others.44,45 KF Determination for G-Quadruplexes Associated with Protein. Nucleic acids are bound by various proteins in vivo and liberated under specific conditions. When a G-quadruplex-forming region is released to fold, its adjacent region may still be associated with proteins that may or stay on and off in response to cellular events. In order to examine how this will affect the folding/ unfolding equilibrium of G-quadruplex, we labeled the G3(T2AG3)3 with digoxin at either one or two ends. Then a 46 kDa antidigoxin antibody Fab fragment was attached to the digoxins. This arrangement is not related to any specific telomere function but to simply mimic physical presence of protein associated besides a G-quadruplex. Four nucleotides were introduced as a spacer between the core sequence and digoxin to avoid potential steric hindrance for the binding of the fluorescent C-rich strand (Figures 3 and 4). The Fab fragment instead of the intact antibody was used to ensure a 1/1 binding stoichiometry between protein and digoxin. When the sequence was attached at one end with a protein, a KF of 156.1 was obtained in 150 mM K+ solution, which is one-fifth of the KF at the free state. A more profound destabilization was observed when an additional protein was attached to the remaining free end. In this case, the KF decreased by 10-fold resulting in a KF of 15.6. This value is more than 50-fold smaller than that of the free core sequence. These results indicate that binding of proteins to one end of a G-quadruplex can significantly destabilize the structure, and restriction at both ends of a G-quadruplex can produce much more significant impact than restriction at one end. DISCUSSION In this work, we introduced an isothermal differential hybridization (IDH) method to measure the folding equilibrium constant KF of G-quadruplexes, which offers several advantages over the conventional thermal melting technique. The measurement is based on the increase in the apparent equilibrium dissociation constant (KDq vs KD, eq 10) of DNA hybridization caused by the formation of G-quadruplex. With the use of a reference hybridization in which the G-rich strand is mutated to prevent G-quadruplex formation to obtain the genuine KD, the KF can then be extracted from two hybridization curves. From Figure 1B, it can be seen that a KF within a wide range of 104 may be properly determined if the KD and KDq is manipulated, by adequate mutations on the C-rich strand, to fall within the range of 10-10 to 10-6 M. Although the extremely high stability makes KF measurement difficult in the thermal melting technique, this turns out to be beneficial in the IDH because it better differentiates the KD and KDq to produce two distinct hybridization curves. (46) Olsen, C. M.; Gmeiner, W. H.; Marky, L. A. J. Phys. Chem. B 2006, 110, 6962–6969. (47) Risitano, A.; Fox, K. R. Biochemistry 2003, 42, 6507–6513.


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Table 1. KD, KDq, and KF for the G3(T2AG3)3 G-Quadruplex Free or Associated with a 46 kDa Anti-Digoxin Antibody Fab Fragment (Ab) at One or Both Ends KD (M) ± SDa

sequence (5′-3′), cation (150 mM) +

G3(T2AG3)3, K

G3(T2AG3)3, Na+ Ab-G3(T2AG3)3, K+ Ab-G3(T2AG3)3-Ab, K+

a

1.015 1.008 1.006 1.140 1.244 1.242 1.074 1.063 9.011 6.131 4.951 6.107

× × × × × × × × × × × ×

KDq (M) ± SDa

-9

-11

10 ± 6.504 × 10 10-9 ± 6.480 × 10-11 10-9 ± 6.475 × 10-11 10-9 ± 7.471 × 10-11 10-9 ± 7.814 × 10-11 10-9 ± 7.805 × 10-11 10-9 ± 5.709 × 10-11 10-9 ± 5.681 × 10-11 10-10 ± 5.230 × 10-11 10-9 ± 4.470 × 10-10 10-9 ± 4.132 × 10-10 10-9 ± 6.495 × 10-10

7.836 8.367 8.282 2.250 2.106 2.183 1.678 1.545 1.529 9.922 8.589 9.861

× × × × × × × × × × × ×

-7

10 10-7 10-7 10-7 10-7 10-7 10-7 10-7 10-7 10-8 10-8 10-8

± ± ± ± ± ± ± ± ± ± ± ±

2.122 2.323 2.290 5.693 5.289 5.503 3.827 3.432 3.386 4.301 3.740 5.727

× × × × × × × × × × × ×

KF -8

10 10-8 10-8 10-9 10-9 10-9 10-9 10-9 10-9 10-9 10-9 10-9

771.0 829.4 822.2 196.5 168.2 174.8 155.3 144.3 168.7 15.18 16.35 15.15

SD represents standard deviation.

Table 2. Comparison of KF of G3(T2AG3)3 and Similar Sequences Obtained with Different Methods at pH 7.0-7.5 at 37 °Ca sequence (5′-3′)

method

G3(T2AG3)3

IDH

G3(T2AG3)3

UV melting

G3(T2AG3)3 G3(T2AG3)3

CD melting CD melting

AG3(T2AG3)3

UV melting

AG3(T2AG3)3 TG3(T2AG3)3

DSC FRET melting

T2AG3(T2AG3)3

CD melting

cation (mM)

Tm (°C)

∆G0 (kcal/mol)

KF

ref

807.5 179.8 3923.0 130.1 19.5 2.68 × 1010 2.0 × 109 2036.0 367.9 130.1 8.31 × 105 560.3 261.3 6.1

this work

65 58 55 69.3 63.7 63 56 66.1 81.8 62.8 63 4.9

-4.13 -3.20 -5.1 -3.0 -1.83 -14.8 -13.2 -4.6 -3.5 -3.0 -8.4 -3.9 -3.4 -1.1

+

K (150) Na+ (150) K+ (100) Na+ (100) Na+ (70) K+ (100) Na+ (100) K+ (100) Na+ (100) K+ (100) K+ (100) Na+ (100) K+ (70) Na+ (70)

43 44 45 43 46 47 44

0

∆G0 in this work was calculated using ∆G0 ) -RT ln(KF). KF from the references was calculated using KF ) e-∆G /RT or ∆G0 ) ∆H0 - T∆S0 where ∆G0 or ∆H0 and ∆S0 were provided. a

Figure 3. Representative measurement of KF for G-quadruplex formed by G3(T2AG3)3 with a 46 kDa Fab fragment of antidigoxin antibody attached to the 5′ end. The KF was obtained from two sets of hybridizations in the same way as in Figure 2. Averaged KF in 150 mM K+ solution was 156.1 from three measurements (Table 1).

As a result, the IDH method should be useful for analyzing highly stable G-quadruplexes. Since an isothermal condition is used, another advantage of the IDH is its applicability to analyze nucleic acids in a physiologically relevant state in the presence of associated proteins or other secondary 9474


Figure 4. Representative measurement of KF for G-quadruplex formed by G3(T2AG3)3 with a 46 kDa Fab fragment of antidigoxin antibody attached to both the 5′ and 3′ end. The KF was obtained from two sets of hybridizations in the same way as in Figure 2. Averaged KF in 150 mM K+ solution was 15.6 from three measurements (Table 1).

structures that comelts with the intended structure in thermal melting. Our measurements revealed information regarding intramolecular G-quadruplex in physiologically relevant structures. The

much smaller KF obtained in the presence of associated protein adjacent to the telomere G-quadruplex indicates that such structures under in vivo conditions are destabilized by adjacent proteins. This property is in agreement with our previous work in which a small KF was obtained for the telomere G-quadruplex restricted by immobilization on solid surface.48 Therefore, it is expected that G-quadruplexes formed in vivo should be much less stable than their free core structures appeared to be in the many in vitro studies. Since stabilization of telomere G-quadruplex by small molecules is emerging as an anticancer strategy,21,49 it is expected that a less stable structure provides more potential for small molecules to stabilize it while a more stable G-quadruplex leaves less room for further stabilization. The destabilization of G-quadruplex should also have implications in molecular machines in which the folding/unfolding cycle of

G-quadruplex is used to provide the driving force,50 since such device needs supporting and has to be interfaced with other parts to operate. A small KF may reflect a decrease in folding rate and is expected to affect the cycling frequency.

(48) Zhao, Y.; Kan, Z. Y.; Zeng, Z. X.; Hao, Y. H.; Chen, H.; Tan, Z. J. Am. Chem. Soc. 2004, 126, 13255–13264. (49) Neidle, S. Curr. Opin. Struct. Biol. 2009, 19, 239–250. (50) Liu, H.; Liu, D. Chem. Commun. (Cambridge, U.K.) 2009, 2625–2636.

Received for review August 18, 2010. Accepted October 15, 2010.

ACKNOWLEDGMENT This work was supported by Grant Numbers 2010CB945300 and 2007CB507402 from MSTC and Grant Numbers 90813031, 30970617, and 20921062 from NSFC. We thank Dr. Di Yu at the Department of Immunology, Monash University, Australia for helping purchase the digibind. SUPPORTING INFORMATION AVAILABLE Additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AC102168M


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Folding Equilibrium Constants of Telomere G-Quadruplexes in Free

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